Microglia differentiation using a culture system for the expansion of mice non-adherent bone marrow stem cells

Abstract

Introduction: Studying primary adult microglia is hampered because of the difficult isolation procedure and the low cell yield. We therefore established a differentiation protocol using a culture system developed for the expansion of non-adherent bone marrow cells.

Methods: Non-adherent bone marrow derived stem cells (NA-BMC) are derived by selective adhesion ('preplating') and are non adhesive adult stem cells. We investigated the changes in bone marrow cell populations by this repeated selective adhesion and compared the potential of the derived cells to differentiate towards microglia. Cells were differentiated with astrocyte conditioned medium (ACM) and granulocyte-monocyte colony stimulating factor (GM-CSF).

Results: NA-BMC cultures show a steep raise in the fraction of stem cells during the cultivation time and the differentiation potential is of the same quality as established protocols. Around 70% of the cells are microglia defined as being positive for CD11b/CD45 and show phagocytosis activity and oxidative bursts.

Conclusion: The non-adherent cell system has the advantage that is produces stem cell progenitors during expansion and provides good microglial differentiation.

Figure 1

(A) Overview showing the two differentiation methods. Representative forward scatter (FSC) and side scatter (SSC) plots of NA-BMC are shown. Day 0 – day 4: Phase of selective adhesion. Day 4 - day 10 and day 1 - day 7: Differentiation phase. (B) Yield of non-adherent cells per whole bone marrow on day 1 – day 4. Repeated selective adhesion results in a falling number of cells.

Figure 2

(A) Scatter plots of NA-BMC during the selective adhesion phase. The indicated regions define populations of immature and nucleated red blood cells, lymphocytes, monocytes and granulocytes. P is the probability of the null hypothesis of the linear regression, i. e. that the percentage fraction does not change over time. (B) Changes in the percentage fraction of the cell populations is shown. Lymphocyte fraction is diminished by selective adhesion. (C) CFU-f grown from NA-BMCs of day 1 to day 4 (n=3). Methylene blue staining was used to determine total colonies. Alizarin red (Calcium), Sirius red (Collagen) and alkaline phosphatase (ALP) staining were used to detect the capacity of NA-BMC for osteogenic differentiation.

Figure 3

Median of NA-BMC antigen expression during day 0 – day 4 of selective adhesion. Both markers of differentiated (F4/80) and undifferentiated hematopoietic cells (CD34) rise. Cell size gets smaller, larger cells seem to become adherent. The regression tests for a statistically significant change over time. P is the probability of the null hypothesis – that marker expression does not change over time.

Figure 4

(A) Representative scatter plots of differentiated cells. A microglia gate was set, defined by the primary microglia population, to assess differentiation. (B) Percent of CD11b/CD45low (microglia gate) among the differentiated cells (n=3). Yields of cells showing the microglia markers are not different between the two protocols. A small subpopulation in fresh bone marrow (10%) shows microglia markers from the beginning. (C) and (D) Percent of CD11b⁺ and CD45⁺ cells among the differentiated cells (n=3). CD45 is already high in fresh bone marrow. P1: Protocol 1. P2: Protocol 2. *** = P ﹤ 0.001, ** = P ﹤ 0.005, * = P ﹤ 0.01.

Figure 5

(A) Representative histogram plots of F4/80 expression. Gray line is the isotype control. (B) Percent of F4/80⁺ cells among the differentiated cells (n=3). Protocol 2 shows higher F4/80 expression than protocol 1 although, during selective adhesion, F4/80 rose steadily. P1: Protocol 1. P2: Protocol 2. *** = P ﹤ 0.001, ** = P ﹤ 0.005, * = P ﹤ 0.01.

Figure 6

(A) Phagocytic activity of differentiated cells. Cells of the classical pre-plating protocol 2 show significantly higher phagocytosis of fluorescent beads. Cells un-supplemented with ACM and GM-CSF show low phagocytosis. (n = 3). (B) Oxidative burst of the differentiated cells is not different between protocol 1 and protocol 2. P1: Protocol 1. P2: Protocol 2. (n = 3). (C-F) Differentiated cells of protocol 1 are smaller and contain many non-adherent cells. Morphologies and resulting cell types are diverse. Images were taken with a Leica DMIL at 200x magnification. (G-J) Most differentiated cells survive in co-culture and remain largely amoeboid and round, typical for activated states. The cells were co-cultured with living brain slices. Top down and side view pictures. Differentiated cells were labeled green (DIO) and transferred onto 9 day old living brain slices. Dead cells were labeled red (propidium iodide). After 10 days of co-culture a Leica Microsystems SP2 confocal microscope was used to scan the brain slices to a depth of 160 μm (Magnification 100x). (H, J) Cells of the cytokine supplemented cultures invade surface up to a depth of 60 μm. *** = P ﹤ 0.001, ** = P ﹤ 0.005, * = P ﹤ 0.01.